Bloodstream trypomastigotes (BSFs) of 2 clones (IL 1392 and IL 3671) of Trypanosoma vivax were cultured without feeder layers in 3 systems. System I: metacyclic-producing cultures of T. vivax IL 1392 were initiated with BSFs derived from infected mice at 27℃ in the presence of feeder layers using TVM-1 medium. Metacyclics harvested were then transferred to flasks containing feeder cells and maintained at 34℃ using TVM-22 medium. Under such conditions, the metacyclics transformed to BSFs. Bloodstream trypomastigotes which were then transferred to new flasks, continued to grow in HMI-162 medium without feeder cells. The maximum density of the BSFs and their shortest population doubling time were 3.5 x l06 / ml and 13.5 h, respectively. System II: metacyclic-producing cultures of T. vivax IL 3671 were initiated with BSFs derived from infected cattle, without feeder layers at 27℃ using HMI-107 medium. Metacyclics were then transferred to the axenic culture conditions established in System I. They also transformed to BSFs and multiplied in HMI-162 and HMI-163 media. System III: cultures were initiated with proboscides of Glossina morsitans centralis which were infected with T. vivax IL 3671, without feeder layers at 34℃ using HMI-162 medium. Metacyclics emerged from the proboscides, transformed to BSFs and continued to grow in HMI-163 medium. The maximum density and the population doubling time of IL 3671 BSFs in HMI-163 medium were 1.8 x 106 / ml and 16.1 h, respectively.
This paper describes a standard method for in vitro excystation of sporocysts of S. capracanis, S. hircicanis, S. ovicanis and S. arieticanis. A standard pretreatment was performed by 20 minutes preincubation of sporocysts in aqueous sodium hypochlorite (NaOCl) solution of 6% or 8% at room temperature. Then, sporocysts were washed 5 times in distilled water and were incubated for one hour at 39℃ in excystation fluid consisting of RPMI 1640 medium, 10% FCS and 15% bovine bile. Additional sonication of pretreated sporocysts increased excystation rates as compared to not sonicated controls. Excystation rates for 1-9 months old sporocysts were 77% for S. capracanis, 77% for S. hircicanis, 72% for S. arieticanis and 92% for S. ovicanis. Sporozoite suspension as obtained by this method proved to be decontaminated and sporozoites were viable and able to invade Vero cells in tissue culture. Sporozoites were also subjected to cryopreservation in RPMI 1640 containing 10% FCS and 7.5% DMSO. After storage in liquid nitrogen, sporozoites proved to be infective for their intermediate hosts after intraperitoneal injection.
The role of pancreatic proteolytic enzymes in the excystation process of Eimeria tenella oocysts and sporocysts was studied in vitro. Intact sporulated oocysts were preincubated in phosphate buffer, NaCl 0.9% (PBS) added with 0.5% chicken bile extract in a 5% CO2 atmosphere for 30 minutes prior to exposure to either 0.25% (w/v) chicken trypsin, chymotrypsin, pancreatic elastase, or a 1% (w/v) crude extract of unsporulated and sporulated oocysts of E. tenella (Expt.1). No excystation was observed under these conditions. Sporocysts were also incubated under the same conditions without pretreatment in CO2. Excystation was observed for sporocysts incubated with either trypsin, chymotrypsin or pancreatic elastase, the best percentage of excystation being recorded for the latter after 5 hours (Expt. 2). Crude extracts of E. tenella oocysts failed to bring about excystation of sporocysts at any time. In experiment 3, sporocysts were incubated with either trypsin, chymotrypsin, or pancreatic elastase alone, any combinations of 2 of these enzymes or with all 3 enzymes. The best percentage of excystation was observed after 5 hours with sporocysts incubated with trypsin and chymotrypsin (99%). The other combinations of 2 enzymes gave also comparable results. Sporocysts incubated in the presence of the 3 enzymes excysted similarly well, although a significantly lower percentage (P<0.05) after 5 hours was recorded when compared to that in sporocysts incubated with trypsin and chymotrypsin. In most cases, the association of 2 or 3 enzymes had a synergistic effect on the percentage excystation of sporocysts in vitro.
A new staining technique of 'thick smears' was developed for diagnosis of malaria using
transmission fluorescence microscopy. Plasmodium falciparum cultured in vitro was used as a model. The infected blood was mixed 1:1 -1:2 with acridine orange (AO) solution (final concentrations of 50-100μg/ml in 0.01 M Tris-HCI buffer or in PBS, pH 7.0～7.5).
Immediately or several minutes later, haemolyzed or non-haemolyzed 'thick smears' were observed directly by fluorescence microscopy using an interference filter specially designed for excitation of AO or commercially available, three (interference-, glass- and triacetyl cellulose film-) types of B-excitation filters with halogen- or daylight-illuminated microscopes. All of these filters were capable of detecting the parasites rapidly in these wet mounts at a magnification of x200 using standard lenses. Among them, the interference-type filters having higher transmission efficiencies gave the best results. Film filter system,
which was the cheapest, could also be used, especially in combination with daylight-illuminated microscopes, although it was necessary to be exposed accurately to the direct rays of the sun. These results strongly suggest that transmission fluorescence microscopy using any B-excitation filter in light microscopes may be a useful economic system for rapid diagnosis of malaria.
Protective immune responses and the functional role of spleen cells in mice infected with
Babesia rodhaini were examined with an in vitro proliferation assay systems and by in vivo passive transfer of spleen cells to uninfected mice. Mice that resolved primary babesial infection after chemotherapy (Babesia immune mice) had transient and low parasitemia after challenge infection and high rates (75%) of survival. Babesia hyperimmune mice, by contrast, had no detectable parasitemia after challenge and 100% survival. Proliferative response of spleen cells to Babesia lysate antigen (BLA) were determined for mice from both groups. This proliferative response was inhibited by treatment of spleen cells with anti-T cell serum and monoclonal antibody (MAb) to Lyt 1 antigen. Spleen cells of hyperimmune mice produced larger amount of IL-2 production than those of immune mice. Transfer of spleen cells from immune mice to nonimmune mice provided protection against babesial infection and recipient mice had high titers of anti-babesial antibody. When these spleen cells were treated with anti-T cell serum or anti-mouse Ig serum, protection against challenge was abolished. By contrast, transfer of hyperimmune spleen cells was capable of protecting recipient mice. Treatment of hyperimmune spleen cells with antiserum to mouse Ig or MAb against Lyt 1 and Lyt 2 antigens did not interfere with their ability to protect recipient mice against infection, even though recipient mice had low levels of antibody
production. These results indicate that humoral immune response is important in establishing protection after primary infections while the participation of Lyt 1+ cells and Lyt 2+ cells and other aspects of the cell-mediated immune response is important in controlling secondary infections.